International Research Journal of Agricultural Science and Soil Science (ISSN: 2251-0044) Vol. 2(11) pp. 469-475, November, 2012 Available online http://www.interesjournals.org/IRJAS Copyright ©2012 International Research Journals Full Length Research Paper Bioactivity of methanolic seed extract of Barringtonia asiatica L. (Kurz) (Lecythidaceae) on biological characters of Spodoptera litura (Fabricius) (Lepidoptera: Noctuidae) Danar Dono, Wahyu D. Natawigena and Mubqi Ghaida Majid Department Plant Pests and Diseases, Faculty of Agriculture, Universitas Padjadjaran, Jatinangor 45363, Indonesia ABSTRACT Plants are known to have various chemical compounds that have potential to be developed as insecticides. One of the potential plants to be developed as a source of insecticides is Barringtonia asiatica (Lecythidaceae). This research was conducted to determine toxicity of methanolic seed extract of Barringtonia asiatica to mortality and biological character of Spodoptera litura. The evaluation of toxicity was carried out using feeding method. Result of this research indicated that methanolic seed extract of B. asiatica had insecticidal activity with LC50 at concentration of 0.30% and LC90 at concentration of 0.80% in 13 days after treatment with LT 50 at 4.8 days. In addition, methanolic seed extract of B. asiatica caused decrease of larval weight, tend to increase duration time of development, reduced leaf consumption and decrease of egg amount oviposited by female of S. litura. Keywords: Barringtonia asiatica, seed, extract, mortality, biological character, Spodoptera litura. INTRODUCTION Various active ingredients originally from plants have been known and tested against insects. At least 2000 plant species have been reported toxic to various plant pests, and more than 850 active compounds from plants have been tested against insects (Grainge and Ahmed, 1988; Prakash and Rao, 1997). During the last decade there is improvement of big enthusiasm in seeking of insecticide compounds from plant (Schmutterer, 1995). Syahputra (2001) reported from various districts in Indonesia, there were more than 40 potential plant species that could be used as botanical insecticides. Botanical insecticides have long been applied by the farmers. One of the plants having a potency to be *Corresponding Author E-mail: [email protected] developed as insecticide is Barringtonia asiatica Kurz (Lecythidaceae) with common name sea poison tree or in Indonesia known as bitung (Ecology and Evolutinonary Biology Greenhouse (EEBG), 2006). B. asiatica is known to have active compounds which cause mortality to insect pests. Methanolic seed extract of B. asiatica was toxic to Crocidolomia pavonana with value of LC50 equal to 0.66% at 7 days after treatment (dat). The application of seed extract of B. asiatica also has influenced oviposition with effective concentration equal to 0.96% which causes C. pavonana female not to lay the eggs at the crop. Larval response indicated that the extract of B. asiatica, besides had toxic character also had antifeedant activity (Dono and Sujana, 2007). Therefore, methanolic seed extract of B. asiatica had a potency as insecticide. One of the active compounds in seed of B. asiatica is saponin (Herltz et al., 2002; Burton et al. 2003, Cannon et al. 2004). At some places B. 470 Int. Res. J. Agric. Sci. Soil Sci. asiatica is used as ancient medicine and fish poison (The Cook Island Nature Heritage Trust, 2005). Active compound in seed of B. asiatica which poisoned fish is group of saponin compounds (Tan, 2002; EEBG, 2006). One of the most toxic compounds against fish from B. asiatica seed extract is ranunkosida VIII (Burton et al, 2003). Research about seed extract of B. asiatica has been conducted but its toxicity to Spodoptera litura(Fabricius) (Lepidoptera: Noctuidae) has not been known. This research was conducted to know the influence of B. asiatica seed extract on mortality and biological characters of S. litura. S. litura is widespread insect, polyphagous, and can eat all parts of crop, especially at dry season (Department of Indonesian Agriculture, 2005). Controlling of S. litura in general is still by using synthetic insecticide. Unfortunately, usage of synthetic insecticides has reported able to generate resistance of insect target, resurgence, and secondary pest out break. Other side impact of usage of synthetic insecticide is environmental contamination and killing of non-target organism (Profit, 1993). Therefore, an alternative insecticide, that was relatively safe for environment and has minimal or no side effect to non target organism, was required. An alternative method for controlling of the pest was botanical insecticide from seed extract of B. asiatica. MATERIALS AND METHODS Rearing of Test Insect Spodoptera litura Test insect larvae, S. litura, was collected from Brassicaceae plantation in Lembang. Larvae obtained from the field were placed in plastic box and feed with taro leaf, and pupation took place in plastic box with sterile soil. Pupae were put in screen cage and adults emerged from pupae was fed with dilution of honey 10% which have been permeated in cotton. In the cage, some leaves and pieces of taro were placed for adult oviposition. Egg mass at taro leaf was removed into separate plastic box. At the time of egg hatching, larvae ware moved to bigger plastic box and fed with taro leaf. Extraction of Seed of Barringtonia asiatica Seeds of B. asiatica was obtained from the field (altitude: 750 m above the sea level) of Campus Universitas Padjadjaran, campus Jatinangor, Sumedang, West Java, Indonesia. Seed was taken away from the fruit, then sliced thin and wind-dried. The dried seeds were refined by using blender to form flour. Flour of the seeds at weight of 2969.34 g was soaked in 10.68 litres of methanol. Methanol was used as solvent because of active compound contain in seed of B. asiatica was polar compound. The solution was filtered using filter paper and evaporated using rotary evaporator at temperature of 40ºC with low pressure until all solvent evaporated and yielded of 781.48 g crude extract. The crude extract was stored in refrigerator at temperature ± 4ºC until being used. Experiment 1. Insecticidal Activity of B. asiatica Against Spodoptera litura The extract was tested against second instar larvae of S. litura using a leaf residue feeding method. Extract was tested at six concentrations that were expected to cause between 0 – 100% insect mortality. The range of concentrations was determined by preliminary test. The treatment with each concentration was replicated six times. Examination was done with taro leaf dipping method. The extract diluted with mixture of aquadestilata with Agristic (0.05%). Two treated or control leaf disks were placed in glass petri dishes (9 cm in diameter) lined with towel paper, and 10 first instar S. litura were placed in each dish. The test larvae were fed with treated leaves for 72 hours, then fed untreated leaves until fourth instar. Parameter observed was mortality of insect. Regression relationship between extract concentration and percentage of insect mortality were determined by probit analysis (Finney, 1971). Calculations of sublethal concentrations of extract were used for other experiments. Experiment 2. The Effect of B. asiatica Seed Extract on Fecundity and Biological Character of Spodoptera litura The treatment procedures were similar with the same as Experiment 1. Concentration of extract tested at this research equivalent with LC30, LC50, and LC70 determined at experiment 1. In each level concentration of test and control larvae was observed until becoming adult. Imagos that were successfully emerged from pupae were paired and then put into different plastic cage (diameter 6.5 cm, height 30 cm). The imago fed with diluted honey (30%) and taro leaf put in plastic container as egg trap. Observation was done every day for larval weight using digital weighting-machine, feed wide eaten, pupa weight, emerge of adult, number of eggs oviposited during the female of adult lived. Larval weight was measured by electrical weighing-machine. While width of food leaf consumed was measured with paper millimeter block. Data obtained were analysed with analysis of variance and continued with multiple range test at significance level of 5%. Dono et al. 471 100 90 80 Control 0.05 % 0.1 % 0.2 % 0.4 % 0.8 % Mortality (%) 70 60 50 40 30 20 10 0 1 2 3 4 5 6 7 8 9 10 11 12 13 Day after treatment (dat) Figure 1. Mortality of S. litura larvae caused by treatment of methanolic seed extract of B. asiatica Table 1. Parameter regression probit relationship between concentration of methanolic seed extract of B. asiatica and mortality of S. litura Day analisys (DA) 3 5 8 10 12 13 a b SE g a ± SE -0.55 ± 0.26 -0.82 ± 0.24 0.32 ± 0.23 0.51 ± 0.23 1.18 ± 0.28 1.12 ± 0.26 b ± SE Value of LC50 0.71 ± 0.38 1.08 ± 0.38 1.27 ± 0.35 1.46 ± 0.36 2.37 ± 0.49 2.15 ± 0.46 (%) 5.89 1.18 0.55 0.44 0.31 0.30 Confidential limits (95%) Value of LC90 Confidential limits (95%) 0.56 – 27.33 0.33 – 1.57 0.29 – 0.88 0.22 – 0.43 0.21 – 0.41 (%) 363. 53 17.99 5.57 3.31 1.09 1.17 3.22 – 47.42 1.82 – 196.3 1.39 – 34.35 0.71 – 2.72 0.74 – 3.05 g 1.01 0.29 0.29 0.23 0.16 0.15 = Intercept = Coefisien of regression = Standard error = Potential index of significance RESULTS AND DISCUSSION Toxicity of Methanolic Seed Extract of Barringtonia asiatica to Spodoptera litura Larvae Mortality of test insect increased equivalently with increasing concentration of methanolic seed extract of B. asiatica applied. In all treatment, larval mortality was seen since 1 day after treatment (dat). Improvement of mortality at concentration of 0.05%, 0.1%, and 0.2% was seen stable at 6 dat, while at concentration of 0.4% and 0.8%, improvement of mortality always happened until 11 dat (Figure 1). Increase of test larval mortality was seen in the first day until 6 DAT. Increasing of test larval mortality after seventh DAT was not significant and at low concentration the larval mortality tend to be stable. The result of research conducted by Dono and Sujana (2007) indicated that methanolic seed extract of B. asiatica tested to Crocidolomia pavonana cause death and inhibit the development of larvae. Bioactivity of active compound in methanolic seed extract of B. asiatica worked slowly with mortality response equal to 90% at concentration of 0.8% at 13 DAT. Saponin is principal active compound in extract of B. asiatica acted as stomach poison so that it doesn't cause death of larva immediately (Agrell et al. 2004). Methanolic seed extract of B. asiatica has insecticidal activity to S. litura larvae. Based on result of lethal concentration (LC50) analysis, the value of LC50 since 3 DATuntil 5 DATwere decreasing and stable at 12 DAT. Value of LC50 obtained at 13 DATwas equivalent with concentration of 0.30% (Table 1). Amount of larval mortality generally occurred at initial instar compared late instar. Difference of level of defence in detoxyfication of poison compound in body causing 472 Int. Res. J. Agric. Sci. Soil Sci. Table 2. Analysis LT50 methanolic seed extract of B. asiatica to S. litura larvae Extract concentration (%) 0.2 0.4 0.8 a ± SE -1.25 ± 0.31 -1.09 ± 0.29 -1.02 ± 0.29 Value of LT50 (days) b ± SE 1.01 ± 0.34 1.11 ± 0.33 1.74 ± 0.34 Confidential limits (95%) 10.02 – 27.07 6.22 – 20.64 3.36 – 4.46 16.9 9.5 4.8 g 0.42 0.33 0.14 a = Intercept b = Coefficient of regression SE = Standard error g = Potential index of significance LT = Lethal time Weight of larvae (g) 0.3 0.25 0.2 Control 0.15 % 0.30 % 0.52 % 0.15 0.1 0.05 0 5 6 7 8 9 10 11 12 13 14 15 Day after treatment (dat) Figure 2. Average of weight of S. litura larvae on each methanolic seed extract of B. asiatica treatment at concentration equivalent with LC30 (0.15%), LC50 (0.30%), and LC70 (0.52%) death response will differ in. According to Syahputra et al. (2006), if larva consumes active compoundm initial instar of larva more sensitive to toxic compound and would easily to die compared to late instar of larva because of the late instar has stronger defence reaction compared to initial instar. Death symptom of larvae showed that initially the larvae were not active,the body looked smaller, seen wrinkled, and then dry. This was indicated that active compound in the form of saponin contain in methanolic seed extract of B. asiatica wasthe main cause of death of larva at all of treatment. According to Chaieb et al. (2001) saponins in the insect body can disturb cell permeability and cause decreased size of the cell because of losing of cell fluid. The breakdown this cell permeability is caused by the reaction between saponins with cholesterol and phospolipid of the cell membrane (Menger and Keiper, 1998). B. asiatica seed extract at lower concentration of extract required more time to cause mortality of test insect compared with height concentration. B. asiatica seed extract at concentration of 0.8% was killing 50% of insect test slowly (LT50 equivalent with 4.8 days), while at concentration of 0.2%, value of LT50 far longer that was 16.9 days (Table 2). Saponin extracted from plant cause death of test insect sufficiently long (Agrell et al. 2004). Influence of Methanolic Seed Extract of B. asiatica to Biological Character of Spodoptera litura Larvae Influence of Methanolic Seed Extract of B. asiatica on Weight of Larvae Larval weight was depended on quantum of food consumed. Decrease in feeding activity caused as a result of toxic compound eaten by larval test can influence growth rate and larval weight (Ambarningrum et al., 2009). This caused difference in improvement of larval weight for every concentration of B. asiatica seed extract tested. Improvement of average of larva weight at control and concentration of 0.15% increasing so closing 0.3 g while at concentration of 0.30% and 0.52%, improvement of average of larva weight still below (under 0.2 g) (Figure 2). Difference of average of larva weight in each concentration showed that at height concentration of methanolic seedextractof B. asiatica can reduce feeding Dono et al. 473 Table 3. Influence of B. asiatica seed extracts to leaf area consumed by S. litura larvae Extract concentration (%) Leaf area consumed at 4 dat (%) (x ± SE)(N) Control 0.15 (LC30) Leaf area consumed at 2 dat (%) (x ± SE) (N) 2.40 ± 0.86 (60) b 1.88 ± 0.36 (60) b 0.30 (LC50) 1.87 ± 0.48 (60) b 12.26 ± 5.51 (40) b 0.52 (LC70) 0.93 ± 0.23 (60) a 5.05 ± 2.05 (38) a 20.75 ± 3.07(60) c 14.30 ± 6.79 (58) b The mean followed by different letter was significance different (α = 0.05) x = average of leaf consumed (mm²) SE = Standard error N = Number of alive larvae dat = day after treatment Table 4. The effect of methanolic seed extract of B. asiatica to developmental time of S. litura larvae (day) Extract Concentration Control 0.15 % 0.30 % 0.52 % Developmental Time Instar I–II (day) (x ± SE) (N) 2.0 2.0 2.0 2.0 ± 0.00 (60) ± 0.00 (60) ± 0.00 (60) ± 0.00 (60) Developmental Time Instar II–III (day) (x ± SE) (N) 1.7 ± 1.64 (60) 1.9 ± 0.40 (46) 2.0 ± 0.22 (33) 2.2 ± 0.44 (26) Developmental Time Instar III-IV (day) (x ± SE) (N) 3.0 ± 0.74 (58) 3.7 ± 0.48 (44) 4.0 ± 0.25 (29) 4.0 ± 0.24 (21) x = Mean of duration of larval development (days) SE = Standard error N = Amount of larvae alive activity of S. litura, causing decreasing of average of larval weight. Toxic compounds contained in seed extract of B. asiatica can influence response of insect physiology (Dadang and Prijono, 2008). Influence of Methanolic Seed Extract of B. asiatica to Leaf Area Consumed by Spodoptera litura Larvae Leaf area consumed by the test larvae indicated smaller with increase of the extract concentration (Table 3). The extract (saponin as main active compound contained in the methanolic seed extract of B. asiatica) indicated to act as antifeedant. Result of Herlt et al. (2002) research indicated that two main saponins isolated from B. asiatica shown anifeedant activity against Epilachna. The same results were indicated by some researchers (Adel et al., 2000; Brown (2006); Szczepanik et al., 2001; Shinoda et al., 2002; Agerbirk et al., 2003; Agrell et al., 2003; Szczepanik et al., 2004). Insect is a heterotroph which must consume other organisms to obtain molecules rich in energy which is needed to its growth and development (Meyer 2005). When the nutrition is not enough, it showed disturbance to metabolic process of the insect which would cause death or abnormal growth and development process. Influence of Methanolic Seed Extract of B. asiatica to Duration of Larval Development Methanolic seed extract of B. asiatica influenced development time of larvae from instar II until instar IV. At higher concentration of B. asiatica seed extract lengthen duration of development time of the test larvae (Table 4). When larvae consumes toxic active compound, some of larvae will death, while another larvae will maximize energy source in the body to eliminate the toxic compound so that the growth and development retarded (Syahputra et al., 2006; Tomia, 2008). Influence of B. asiatica Seed Extract to Pupal Weight and Emergence of S. litura adult B. asiatica seed extract influenced pupal formation and weight, and adult emergence (Table 5). Pupal formation at treatment was smaller than control pupae. This indicated that nutrition supplay annoyed at the larval stage. The growth of larvae becomes pursued and finally will influence development of S. litura larvae when forming pupa. Forming of imperfect pupa can reduce emergence of insect adult. There was failure in pupal formation at treatment with 474 Int. Res. J. Agric. Sci. Soil Sci. Table 5. Effect of B. asiatica seed extract on pupal forming and weight, adult emergence of S. litura Treatment Control 0.15 % (LC30) Pupal forming (%) 39.65 39.53 Pupal Weight ± (SE) (g) (N) 0.28 ± 0.34 (20) 0.28 ± 0.23 (17) Adult emergence (%) 0.69 0.53 0.30 % (LC50) 35.71 0.26 ± 0.21 (15) 0.52 0.52 % (LC70) 33.33 0.25 ± 0.17 (11) 0.45 SE = Standard Error N = Number of insect test g = gram Table 6. Amount of egg laid by adult of S. litura emerged from test insect treated with methanolic seed extract of B. asiatica in larval period Extract concentration (%) Control 0.15 (LC30) Amount of eggs (x ± SE) 1343± 0.22 a 1236± 0.12 a 0.30 (LC50) 1172 ± 0.42 a 0.52 (LC70) 1138 ± 0.15 a x = Number of eggs reproduced SE = Standard error higher concentration of extract. It indicated the lack of nutrition supply to S. litura larvae as resulted inhibition of insect feeding activity by active compound of B. asiatica seed extract. In addition, failure in pupal formation could be due to decreasing of food consumption by the insect as a result of toxic compound (Adel et al., 2000). The treatment with concentration of 0.15%, the pupa that was formed successfully, equal to 39.53% with emergence of adult equal to 0.53%, while at concentration of 0.52%, pupal formed only equal to 33.33% with emergence of adult only 0.45%. Pupa that was not formed perfectly at concentration of0.52% caused decreasing of adult emergence. The data indicated that there was decreasing activity of methanolic seed extract of B. asiatica which in line with decreasing of extract concentration. Influence of Methanolic Seed Extract of B. asiatica to Fecundity of Spodoptera litura Female Number of eggs yielded at all of treatments did not different (Table 6). This indicated that active compound in methanolic seed extract of B. asiatica did not caused decrease of reproduction level of S. litura with indicated that most of toxic compound that successfully enters into larval body was successfully neutralized when caterpillar grows from initial instar towards final instar in parallel with moulting process of insect. The value followed by the same letter not significantly different (Duncan test at α= 0.05) The findings above increasingly strengthens estimation that active compound activity coming into larval body of S.litura as a result of treatment of methanolic seed extract of B. asiatica will decline and disappear in line with growth and development from larval stadium, pupa and then adult. Defence mechanism in insect body will try to eliminate toxic compound coming into the insect body and cause imago to successfully emerge from pupa will return to normal growth and can lay eggs again normally. CONCLUSION Result of this research indicated that methanolic seed extract of B. asiatica had insecticidal activity with LC50 at concentration of 0.30% and LC90 at concentration of 0.80% in 13 days after treatment with LT50 at 4.8 days. 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